Method and device for capsulating microbial, plant and animal cells or biological and chemical substances

ABSTRACT

The invention relates to a method for capsulating microbial, plant and animal cells or biological and chemical substances, using a nozzle to obtain small, especially spherical particles by vibrating an immobilisation mixture. According to said method, the immobilisation mixture, especially a laminar fluid jet taking the form of an immobilisation mixture, is divided into equal parts by superimposition of an external vibration. In a device especially well suited to carry out this method a metallic counter-element ( 18 ) which is mounted down-stream from the nozzle ( 16 ) at a distance (a) to, and on the outside of, the nozzle axis (A) is connected to a high-voltage source ( 30 ). The counter-element is to be embodied by a metal ring ( 18 ) through whose through hole ( 20 ) the nozzle axis (A) extends. The metal ring ( 18 ) is radially connected to an insulated support ( 22, 24 ).

The invention concerns a method and an apparatus for encapsulatingmicrobial, vegetable and animal cells or biological and chemicalsubstances through a nozzle to obtain small, substantially sphericalparticles.

The encapsulation of microbial, vegetable and animal cells andbiological and chemical substances—such as catalysts—is of greatsignificance in particular in biotechnology and medicine forimmobilisation purposes. In medicine, encapsulation additionally servesto provide a screening effect from the immune system. By virtue of theimmobilisation effect, it is possible for the cells or the catalyst tobe retained in the process and for the product to be harvested at thesame time. That permits use over a prolonged period of time and affordsan increased space-time yield. By virtue of the cells being screenedfrom the immune system, it is possible to implant in a patient cellswhich are foreign to the body and which over a relatively long period oftime discharge a desired substance into the body of the patient withoutthe cells being attacked and destroyed by the patient's immune system.

Encapsulation of cells and catalysts in biopolymers—such as carrageenanor alginate—and synthetic polymers—such as polyacrylamide—is a methodwhich has been used for some years in research laboratory situations.Many different apparatuses are described for that purpose in theliterature. One of the most efficient methods involves dividing up a jetby the superimposition of an external vibration on the immobilisationfluid. The fluid is thereby divided into fractions of equal size as itissues in a laminar flow from a nozzle. A number of methods for thetransmission of vibration are used or described, for example coupling toa vibrator, piezoelectric crystal, sound waves.

WO 96/28247 to the present applicants discloses a commercialencapsulation unit in which the vibration is transmitted by a rigidconnection to a vibrator. That method suffers from the difficulty thatthe axis of the vibrator and the axis of the nozzle have to be exactlyaligned as otherwise disturbances occur, which massively adverselyaffect the homogeneity of the sphere size. The vibrator is alsoexpensive. In addition, it has been found by photographic analysisprocedures and observations under stroboscope light that, in regularoperation of the apparatus, a monodisperse and single-strand chain ofspheres is visible to about 100 mm downstream of the nozzle. If thespheres are caught after about 100 mm dropping distance in a hardeningbath and are thereafter examined under a microscope, then very oftenbatches without a monodisperse group of spheres is obtained—and this wasnot predictable. The samples generally had three different spherepopulations in a varying ratio; the first was of the expected spherediameter, the second was of double or a multiple greater volume thanexpected, and the third was in the form of two individual spheres whichtouch each other to a greater or lesser degree.

In consideration of that state of the art the inventor set himself theaim of optimising an apparatus and a method of the kind set forth in theopening part of this specification.

That object is attained by the teachings of the independent claims; theappendant claims set forth advantageous developments. In addition thescope of the invention embraces all combinations of at least two of thefeatures disclosed in the description, the drawing and/or the claims.

In accordance with the method according to the invention theimmobilisation mixture, in particular a laminar fluid jet, is separatedinto parts of equal size by the superimposition of an externalvibration. An electrical field is built up in the proximity of thenozzle so that an electrical charge flux occurs in the fluid jet,whereby the drops produced have an electrical charge. That charge mustbe so high that the spheres mutually repel because of the similar chargeand the chain of spheres which is initially present in the form of asingle strand is divided into many partial chains. For that purpose,voltages are required which are preferably in the range of between 200and 1600 V. Due to the dispersing effect, the spheres no longer drop ona closely defined region on to the surface of the hardening bath, butthey are scattered far and wide.

In that way it is now possible as a routine matter to obtain amonodisperse sphere array not only in the air but also in the hardeningbath. Likewise, in the case of immobilisation mixtures which by virtueof their chemical and physical properties could be scarcely or onlypartially put into drop form, it is now also often possible to achieve amonodisperse sphere assembly.

An apparatus which is intended for that method is distinguished interalia in that a metal counterpart element which is arranged downstream ofthe nozzle at a spacing and outside the nozzle axis is connected to ahigh-voltage source. That counterpart element is preferably in the formof a metal ring having an aperture through which the nozzle axis was topass. Provided between the nozzle and the counterpart element or metalring is an electrical field, preferably with the above-mentioned voltagerange.

It has also proven to be advantageous, when dividing the immobilisationmixture by the superimposition of an external vibration into fractionsof equal size, for those vibrations to be transmitted to theimmobilisation mixture either within a pulsation space or chamber or byway of the nozzle which is caused to pulsate. Provided for that purposeis an apparatus in which a pulsation chamber which is arranged upstreamof the nozzle and which receives the immobilisation mixture has apermanent magnet superimposed thereon and the permanent magnet isarranged opposite an electrical coil; in accordance with the inventionone of the two units is provided within the pulsation chamber or on adiaphragm which extends over the pulsation chamber, while the other unitis separated by an air gap from that which is associated with thepulsation chamber.

In another embodiment of the apparatus the permanent magnet and theelectrical coil are associated with the nozzle or the suspension thereofso that same can initiate the pulsation procedure.

The principle of the vibrator comprising the magnet and a coil throughwhich alternating current flows is taken from the vibrator, and a partthereof is directly associated with the pulsation chamber. Whenalternating current is passed through the coil, it is alternatelymagnetised positively and negatively. The magnetic waves interact withthe subjacent magnet and cause it to vibrate. The vibrations aretransmitted almost without resistance to the immobilisation fluid.

In accordance with a further feature of the invention the coil throughwhich alternating current flows and the permanent magnet producevibrations in the preferred range of between 300 and 4000 Hz.

Thus using simple means the invention permits miniaturisation ofvibration transmission, with a very low level of expenditure in terms ofmaterial and energy. The costs of the method and the apparatus can bereduced by a multiple in comparison with the previously known vibrationmethods. A further advantage to be considered here is that theorientation of the magnet and the coil does not have to be centered toan accuracy of 0.1 mm. There are also no axes which have to be preciselyoriented.

Further advantages, features and details of the invention will beapparent from the description hereinafter of preferred embodiments andwith reference to the drawing in which:

FIG. 1 is a side view of an apparatus according to the invention,

FIG. 2 is a perspective view of another apparatus according to theinvention,

FIG. 3 is a plan view of the partly sectioned apparatus in FIG. 2, and

FIG. 4 is a view in section through FIG. 3 taken along line IV—IVtherein.

In an installation of which only part is shown for the sterileencapsulation of microbial, vegetable and animal cells, disposedhorizontally in a reactor 10 above a hardening bath 12 and below anozzle 16 which is suspended from a reactor cover 14 and at a spacing ain relation to the nozzle 16 is a metal ring 18 having a centralaperture 20 through which the nozzle axis A passes.

The metal ring 18 is secured by means of a radial holder 22 and a tube24 connected thereto in an insulating connection portion 26 in thereactor cover 14 and is connected through a line 28 disposed in the tube26 to a high-voltage source 30.

An encapsulation mixture comprising an immobilisation matrix and cellsor substances is conveyed through the nozzle 16 in such a way that afree laminar jet is produced. By virtue of a vibration beingsuperimposed on the free jet, the free jet is broken up into drops K ofequal size. When the fluid penetrates into an electrical field which isbuilt up between the metal ring 18 and the nozzle 16, a charge fluxoccurs in the direction of the nozzle 16 so that the separated drops Khave an electrical charge, being an electrostatic charge. That similarcharge causes mutual repulsion of the drops K.

That procedure results in two effects. On the one hand, the drops K arestabilised in the axial direction, that is to say as soon as two drops Kcome closer together by virtue of different speeds of fall, they arerepelled by the coulomb forces and they cannot come into contact witheach other. On the other hand, very small radial displacements areincreased and the single-strand chain of spheres is expanded to form acone Q. Due to that effect, coagulation of drops K is practicallyprevented and particles of completely equal size are produced in thehardening bath 12. The charges are removed by grounding of the hardeningbath 12 at 32.

In an embodiment of a further installation for sterile encapsulation ofmicrobial, vegetable and animal cells, arranged above the hardening bath12 is a carrier plate 40 of a thickness b, which for example isrectangular, with a depression 44 of a depth t which is formed in thecenter of its surface 42; the depth t corresponds approximately to onethird of the plate thickness b.

The depression 44, as shown in FIG. 4, is defined by a circularperipheral wall 46 of a diameter d, and a bore 50 extends from thecenter point of its bottom 48. The bore 50 opens at the other end in acup-shaped recess 52 of a diameter d₁ (approximately one third of d),which is provided in the underneath surface 41 of the carrier plate 40and in which is carried a nozzle 54 connected to the bore 50. Inaddition, in the plane of the bottom 48, a radial passage 56 leads to alateral blind hole 58 for a connecting portion 60.

Associated with the depression is a pressure ring 66 which is fixed onthe plate surface 42 with the interposition of a diaphragm 62 and a seal64; the pressure ring 66, like also the seal 64, is provided with aninternal aperture 68 of a diameter d and the diaphragm 62 which carriesa disk magnet 70 extends over the depression 44. The diameter e of themagnet 70 is somewhat greater than the diameter d₁ of the recess 52 forthe nozzle 54.

An electrical coil 74 is suspended from a holder 72 at a spacing withrespect to the disk magnet 70, centered with respect to the center lineM thereof. The disk magnet 70 and the coil 74 through which alternatingcurrent flows form a vibrator; when alternating current is passedthrough the coil 74 it is alternately positively and negativelymagnetised. The magnetic waves act on the subjacent disk magnet 70 andcause it to vibrate together with the diaphragm 62.

An immobilisation fluid is introduced through the radial passage 56 intothe depression 44 which forms a pulsation chamber, the vibrations beingtransmitted almost without resistance to the immobilisation fluid. Theintroduction of that immobilisation mixture is effected by means of amechanical feed thrust or by air pressure into the pulsation chamber orrecess 44; from there the immobilisation mixture is urged through thenozzle 54. The jet E which is produced there, shortly after issuing fromthe nozzle 54, breaks up into spheres K₁ of equal size, according to thefrequency of the superimposed vibration. At about 700 Hz, under optimumconditions, 700 of the equal-sized spheres K₁ are produced per second,while the homogeneity of the sphere configuration is excellent by virtueof the friction-less transmission. Measurements have shown that thepower required is less than 0.2 W.

In an embodiment not shown herein the permanent magnet 70 or the coil 74is provided directly at the nozzle 54 and the respective other unit isassociated therewith, forming an air gap.

What is claimed is:
 1. A method for encapsulating an immobilizationmixture comprising the steps of: providing a stream of an immobilizationmixture; vibrating the stream wherein the stream is formed into drops bythe vibration; subjecting the drops to a magnetic fluid wherein thedrops are electrically charged thereby resulting in mutual repulsion;and passing the charged drops to a hardening bath for encapsulating. 2.A method as set forth in claim 1, wherein immobilization mixture isdivided into fractions of equal size by the vibration.
 3. A method asset forth in claim 1, wherein the vibration is transmitted to theimmobilization mixture within a pulsation chamber.
 4. A method as setforth in claim 1, wherein vibration is transmitted to the immobilizationmixture by way of the nozzle which is caused to pulsate.
 5. A method asset forth in claim 1, wherein the vibration is in the range of between300 and 4000 Hz.
 6. A method as set forth in claim 1, wherein the dropsform a cone beneath the magnetic field and above the hardening bath. 7.A method as set forth in claim 1, wherein the electrical voltage of themagnetic field is in the range of between 200 and 1600 V.
 8. A methodfor encapsulating an immobilization mixture comprising the steps of:providing a nozzle; providing a ring having an aperture in line with thenozzle downstream of the nozzle at a distance “a”; providing a magneticfield between the nozzle and the ring; vibrating the ring; and passingthe immobilization mixture from the nozzle, through the magnetic fieldand the aperture in the vibrating ring whereby the immobilizationmixture is formed by vibration into drops having an electrical chargewhich results into mutual repulsion of the drops.
 9. An apparatus forencapsulating an immobilization mixture comprises: a nozzle having anoutlet; a metal ring positioned beneath the nozzle a distance “a”, themetal ring having an aperture in line with the outlet of the nozzle; anda high voltage source connected to the metal ring.
 10. An apparatus asset forth in claim 9, wherein the high voltage source creates anelectrical field between the nozzle and the metal ring of an electricalvoltage in the range of between 200 and 1600 V.
 11. An apparatus as setforth in claim 9, wherein the metal ring is radially connected to aninsulatedly mounted holder.
 12. An apparatus as set forth in claim 9,wherein a pulsation chamber is arranged upstream of the nozzle andreceives the immobilization mixture, a permanent magnet acts on thepulsation chamber and is arranged opposite an electrical coil.